The use and applications of Cd-based quantum dots (QDs) have increased in the past few years. Some desirable features of these nanostructures include their narrow emission spectra, large quantum yield, chemical stability against photo-bleaching, emission spectrum in the visible range and easy surface functionality. These characteristics enable the use of these QDs in biological staining, detection of bio-macromolecules, disease diagnostics, among others. Water-stable Cd-based QDs functionalized with organic materials have shown good chemical stability without affecting their optical properties. The functionalization of QDs using aminoacids, peptides (like glutathione) and proteins assures the water stability, biocompatibility and hence their potential biological applications.
The microwave-assisted synthesis of water-stable Cd-based QDs in the presence of thiol reagents represents a fast and one-step synthesis approach with less generation of hazardous waste.
Although the water-stable QDs with good optical properties synthesized using the microwave irradiation technique can find potential applications in nanotechnology, as-synthesized QDs will still require rigorous purification and control of the crystal size at the nanoscale. Accordingly, the development of a size-exclusive protocol with capability to purify and generate size-selected fractions from a suspension of nanocrystals becomes indispensable.
High performance size exclusion chromatography (HPSEC) has been used as a powerful tool to separate complex samples based on their sizes. HPSEC applications have been expanded to the characterization and separation of nanomaterials as semiconductor nanocrystals or quantum dots (QDs). The separation by HPSEC is based on purely entropic interactions between the pores of the polymeric material in the column and the nanomaterials while avoiding all other interactions such as polar-polar, nonpolar-nonpolar, electrostatic, among others. The unwanted interaction between the surface of functionalized Cd-based QDs and the polymeric material in the column would affect the precision of the correlation between the retention time and the hydrodynamic diameter or the molecular weight of the crystals.
HPSEC overcomes many of the limitations of other techniques used for the size-sensitive separation of nanoparticles. HPSEC has the advantage of being able to measure the hydrodynamic diameter of the nanoparticles suspended in an aqueous solution; this diameter includes the inorganic core and the organic coating (capping agent) of the QDs surface. Regarding the measurement of the actual nanocrystal size, previous related studies reported that Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) techniques fail in the precise determination of the size in extremely small nanocrystals. In contrast, HPSEC is an efficient technique to determine the actual size of nanocrystals, particularly in the 0.5-10 nm range. Furthermore, HPSEC measurement includes millions of nanoparticles making the results statistically robust. Besides, HPSEC can also be considered a semi-preparative technique, which allows samples to be simultaneously separated, quantified and collected for further assays. Despite the promising application of SEC-based techniques, there are still few reports concerning their application in the analysis and separation of nanoparticles. Available literature indicates that the HPSEC-based separation of nanocrystals stabilized in organic solvents such as toluene is possible: Wilcoxon et al. used SEC to separate non-polar gold and silver nanoparticles; Krueger et al. separated nanocrystalline CdSe. Al-Somali et al. separated and analyzed a series of gold nanocluster fractions. Wang et al. isolated QDs of CdSe with dye labeled multidentate polymers. Apparently, nanocrystals soluble in organic solvents did not interact with the polymeric matrix of the columns, which allowed the use of commercially available columns. Also, the mentioned reports used organic solvents as the mobile phase. On the other hand, the use of commercial HPSEC columns for the separation of water-stable nanocrystals becomes troublesome due to the strong interactions between the nanocrystals and the polymeric matrix of the columns. Some authors assembled their own HPSEC columns using specific polymeric matrixes which allow the separation of specific nanocrystals. Arita et al. used nanoparticle repelling surfaces (concentrated polymer brush) to separate QDs and nanoparticles according to their sizes. In some cases the capping of nanocrystals with peptides or proteins also allowed the use of commercial columns designed to separate these kinds of molecules. Ding et al. used HPSEC to separate aqueous solution of CdSe functionalized with proteins using a commercial column. The limiting factor to the broad use of SEC-related approaches in aqueous medium relies on the customized selection and assembly of the stationary phase (column) to achieve the size-sensitive separation of targeted quantum dots.